Toshifumi Hatta

Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.

Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides

We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase‐associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells. Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2–as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.

In Vitro and In Vivo Anti-influenza A Virus Activity of Antisense Oligonucleotides

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.

Inhibitory Effect of Modified 5′-Capped Short RNA Fragments on Influenza Virus RNA Polymerase Gene Expression

We have shown previously that the 5′-capped short phosphodiester RNA fragments, Cap decoy, (Gm 12 nt) are potent inhibitors of influenza virus RNA polymerase gene expression. Here we investigate the modified capped RNA derivative containing phosphorothioate oligonucleotides (Cap decoy) as a potential influenza virus RNA polymerase inhibitor. The modified 5′-capped short phosphorothioate RNA fragments (Gms 12–15 nt) with the 5′-capped structure (m7GpppGm) were synthesized by T7 RNA polymerase. The 5′-capped short RNA fragments (Gms 12–15 nt) were encapsulated in liposome particulates and tested for their inhibitory effects on influenza virus RNA polymerase gene expression in the clone 76 cells. The 12–15 nt long Gms RNA fragments showed highly inhibitory effects. By contrast, the inhibitory effects of the 13 nt long short RNA fragments (Gm 13 nt) were considerably less in comparison with the 5′-capped short phosphorothioate RNA fragments (Gms 12–15 nt). In particular, the various Gms RNA chain lengths showed no significant differences in the inhibition of influenza virus RNA polymerase gene expression. Furthermore, the capped RNA with a phosphorothioate backbone was resistant to nuclease activity. These phosphorothioate RNA fragments exhibited higher inhibitory activity than the 5′-capped short RNA fragments (Gm 12 nt). These decoys may prove to be useful in anti-influenza virus therapeutics.

Binding Modes of Two Novel Non-Nucleoside Reverse Transcriptase Inhibitors, YM-215389 and YM-228855, to HIV Type-1 Reverse Transcriptase

Background: YM-215389 and YM-228855 are thiazolidenebenzenesulfonamide (TBS) derivatives and novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) that inhibit not only wild-type, but also the K103N- and Y181C-substituted reverse transcriptase (RT) of HIV type-1 (HIV-1). Methods: To characterize the binding modes of the TBS derivatives in detail, the anti-HIV-1 activities of YM-215389 and YM-228855 against various NNRTI-resistant clones were examined. Docking studies with HIV-1 RT were also performed. Results: YM-215389, which effectively inhibits various NNRTI-resistant clones, interacted with L100, K103, V106 and Y318 through the benzene ring and with E138, V179, Y181, Y188 and W229 through the thiazole ring. A single amino acid substitution confers only moderate resistance to YM-215389; indeed, four amino acid substitutions (V106L, V108I, E138K and L214F) were necessary for high- level resistance. Although the activity of YM-228855, a derivative of YM-215389 that has two bulky and rigid cyano-moieties on the benzene ring, was 10x more potent against HIV-1 than YM-215389, its anti-HIV-1 activity was readily reduced with single substitutions as with Y181I and K103N. Conclusions: These results provide structural information for optimizing the TBS derivatives in an attempt to construct ideal NNRTIs that maintain anti-HIV-1 activity to various HIV-1 variants.

Inhibition of Reverse Transcriptase-Mediated cDNA Synthesis by Antisense Oligonucleotides

Abstract Unmodified and modified (phosphorothioate) oligonucleotides inhibit cDNA synthesis by reverse transcriptase (RT). Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. However, phosphorothioate oligomer, inhibit polymerization by binding to the AMV RT or MMLV RT, rather than to the template RNA. There was no competitive binding of the phosphorothioate oligomer to the HIV RT during reverse transcription.

ANTI-INFLUENZA VIRUS ACTIVITIES OF NICKED AND CIRCULAR DUMBBELL RNA/DNA CHIMERIC OLIGONUCLEOTIDES

Abstract We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.